Bisulfite-sequencing analysis of previously reported m5C sites in HIV RNA. (A) Schematic representation of HIV genome structure. Amplicon sequences harboring seven previously reported m5C candidate sites (marked in red) in the gag-pol region (left; Cristinelli et al. 2021) and amplicon sequence covering previously reported meRIP peaks (right; Courtney et al. 2019b) are shown (numbers corresponding to AF324493.2 sequence). (B) Bisulfite-treated RNA from virus-infected PM1 cells was reverse transcribed, and the regions indicated in (A) were amplified by PCR followed by subcloning and Sanger sequencing of individual clones (n = 35). No unconverted Cs were detected at the indicated positions in viral RNA. For amplicon 9055–9256, no unconverted C was detected over the whole range. Methylated positions C38, C47, and C48 in human tRNAAsp are shown as positive control (n = 20); results from the spike-in ERCC136 RNA (nt 81–323) are shown as negative control (n = 10).
